Measurement of microbiological activity in an opaque medium

A process for monitoring of microbiological populations in an opaque medium comprising:

• (A) separating at least two Aliquots of material, optionally three Aliquots of material, from the opaque medium;
• (B) adding nothing to the first Aliquot, wherein said first Aliquot is now referred to as Aliquot-Blank, adding a Fluorogenic Dye to the second Aliquot, wherein said second Aliquot is now referred to as Aliquot-Dye, and when the optional third Aliquot is present, adding a Metabolic Inhibitor to the optional third Aliquot, followed by adding Fluorogenic Dye to the optional third Aliquot, wherein said third Aliquot is now referred to as optional Aliquot-Inhibitor-Dye;
• (C) allowing said Fluorogenic Dye to react with any microbiological organisms present;
• (D) providing means for measurement of the fluorescent signals in said Aliquot-Blank, in said Aliquot-Dye, and in said optional Aliquot-Inhibitor-Dye, with the fluorescent signals being measured at the wavelength of the Fluorogenic Dye and at the wavelength of the Reacted Fluorogenic Dye;
• (E) using said means for measurement of said fluorescent signals to measure the fluorescent signals in Aliquot-Blank, Aliquot-Dye, and in optional Aliquot-Inhibitor-Dye, at the wavelength of the Fluorogenic Dye and at the wavelength of the Reacted Fluorogenic Dye, while discarding any measured fluorescent signal values below a predetermined noise level;
• (F) calculating the Useful RATIO, wherein the Useful RATIO is selected from the group consisting of RATIO of Adjusted for Background Fluorescence Fluorescent Signal of the Reacted Fluorogenic Dye to the Adjusted for Background Fluorescence Fluorescent Signal of the Fluorogenic Dye and RATIO of the Adjusted for Interactions with chemicals and Background Fluorescence Fluorescent Signal of the Reacted Fluorogenic Dye to the Adjusted for Interactions with chemicals and Background Fluorescence Fluorescent Signal of the Fluorogenic Dye;
• (G) using the Useful RATIO to monitor the extent of microbiological contamination in said opaque medium.

A process for monitoring of microbiological populations in an opaque medium comprising:

• a) obtaining an Aliquot of material from the opaque medium;
• b) adding a Fluorogenic Dye into said Aliquot, wherein said Aliquot is now referred to as Aliquot-Dye;
• c) allowing said Fluorogenic Dye to react with any microbiological organisms present for a time period known as Time Zero;
• d) providing means for measurement of the fluorescent signals of the Fluorogenic Dye and the Reacted Fluorogenic Dye in said Aliquot-Dye;
• e) using said means for measurement of said fluorescent signals to measure the fluorescent signals of the Fluorogenic Dye and the Reacted Fluorogenic Dye at Time Zero, while discarding any measured fluorescent signals values below a predetermined noise level;
• f) calculating the RATIO of the fluorescent signal of the Reacted Fluorogenic Dye to the fluorescent signal of the Fluorogenic Dye and designating that RATIO the RATIO at Time Zero;
• g) waiting for a time period, designated Time Future;
• h) measuring the fluorescent signals of the Fluorogenic Dye and the Reacted Fluorogenic Dye in Aliquot-Dye at Time Future;
• i) calculating the RATIO of the fluorescent signal of the Reacted Fluorogenic Dye at Time Future to the fluorescent signal of the Fluorogenic Dye at Time Future, designating that RATIO the RATIO at Time Future;
• j) comparing the RATIO at Time Future to the RATIO at Time Zero;
• k) using the comparison of the RATIO at Time Future to the RATIO at Time Zero to monitor the extent of microbiological contamination in said opaque medium.

A process for monitoring of microbiological populations in an opaque medium comprising:

• (A) separating at least two Aliquots of material, optionally three Aliquots of material, from the opaque medium;
• (B) adding nothing to the first Aliquot, wherein said first Aliquot is now referred to as Aliquot-Blank, adding a Fluorogenic Dye to the second Aliquot, wherein said second Aliquot is now referred to as Aliquot-Dye, and when the optional third Aliquot is present, adding a Metabolic Inhibitor followed by a Fluorogenic Dye to the optional third Aliquot, wherein the optional third Aliquot is now referred to as optional Aliquot-Inhibitor-Dye;
• (C) allowing said Fluorogenic Dye to react with any microbiological organisms present for a time period known as Time Zero;
• (D) providing means for measurement of the fluorescent signals in said Aliquot-Blank, in said Aliquot-Dye and in said optional Aliquot-Inhibitor-Dye, with the fluorescent signals being measured at the wavelength of the Fluorogenic Dye and at the wavelength of the Reacted Fluorogenic Dye;
• (E) using said means for measurement of said fluorescent signals to measure the fluorescent signals in Aliquot-Blank, Aliquot-Dye and in optional Aliquot-Inhibitor-Dye at Time Zero, at the wavelength of the Fluorogenic Dye and the wavelength of the Reacted Fluorogenic Dye, while discarding any measured fluorescent signal values below a predetermined noise level to yield fluorescent signals at Time Zero;
• (F) calculating the Useful RATIO at Time Zero, wherein the Useful RATIO at Time Zero is selected from the group consisting of RATIO at Time Zero of the Adjusted for Background Fluorescence Fluorescent Signal of the Reacted Fluorogenic Dye to the Adjusted for Background Fluorescence Fluorescent Signal of the Fluorogenic Dye at Time Zero and optional RATIO of the Adjusted for Interactions with chemicals and Background Fluorescence Fluorescent Signal of the Reacted Fluorogenic Dye to the Adjusted for Interactions with chemicals and Background Fluorescence Fluorescent Signal of the Fluorogenic Dye;
• (G) waiting for a time period, designated Time Future;
• (H) using said means for measurement to measure the fluorescent signals at Time Future in Aliquot-Blank, Aliquot-Dye and in optional Aliquot-Inhibitor-Dye at the wavelength of the Fluorogenic Dye and the wavelength of the Reacted Fluorogenic Dye;
• (I) calculating the Useful RATIO at Time Future, wherein the Useful RATIO at Time Future is selected from the group consisting of RATIO at Time Future of the Adjusted for Background Fluorescence Fluorescent Signal of the Reacted Fluorogenic Dye to the Adjusted for Background Fluorescence Fluorescent Signal of the Fluorogenic Dye at Time Future and optional RATIO at Time Future of the Adjusted for Interactions with chemicals and Background Fluorescence Fluorescent Signal of the Reacted Fluorogenic Dye to the Adjusted for Interactions with chemicals and Background Fluorescence Fluorescent Signal of the Fluorogenic Dye;
• (J) comparing the Useful RATIO at Time Future to the RATIO at Time Zero;
• and
• (K) using the comparison of the Useful RATIO at Time Future to the RATIO at Time Zero to monitor the extent of microbiological contamination in said opaque medium.

A process for monitoring both active and inactive microbiological populations in an opaque medium, optionally accounting for chemical interference with the test method, as well as optionally accounting for background fluorescence comprising:

• (A) obtaining two Aliquots of material, optionally three or four Aliquots of material from the opaque medium;
• (B) adding a Fluorogenic Dye directly into the first Aliquot, wherein the first Aliquot is now referred to as Aliquot-Dye, adding Nutrient and Fluorogenic Dye to the second Aliquot, wherein the second Aliquot is now referred to as Aliquot-Nutrient-Dye, when the optional third Aliquot is present, adding a Metabolic Inhibitor and Fluorogenic Dye to the optional third Aliquot, wherein the optional third Aliquot is now referred to as optional Aliquot-Inhibitor-Dye, and when the optional fourth Aliquot is present, adding nothing to the fourth Aliquot, wherein the fourth Aliquot is now referred to as optional Aliquot-Blank;
• (C) allowing said Fluorogenic Dye to react with any microbiological organisms present for a time period known as Time Zero;
• (D) providing means for measurement of the fluorescent signals in said Aliquot-Dye, said Aliquot-Nutrient-Dye, said optional Aliquot-Inhibitor-Dye and in said optional Aliquot-Blank, with the fluorescent signals in each Aliquot being measured at the wavelength of the Fluorogenic Dye and the wavelength of the Reacted Fluorogenic Dye;
• (E) using said means for measurement of said fluorescent signals to measure the fluorescent signals at Time Zero in said Aliquot-Dye, said Aliquot-Nutrient-Dye, said optional Aliquot-Inhibitor-Dye and in said optional Aliquot-Blank, at the wavelength of the Fluorogenic Dye and at the wavelength of the Reacted Fluorogenic Dye to yield fluorescent signals at Time Zero;
• (F) calculating the Useful RATIO at Time Zero, wherein the Useful RATIO at Time Zero can be selected from the group consisting of RATIO at Time Zero of the Total Microbiological, Optionally Accounting for Interactions with chemicals and Optionally Accounting for Background Interferences Fluorescent Signal of the Reacted Fluorogenic Dye to the Total Microbiological, Optionally Accounting for Interactions with chemicals and Optionally Accounting for Background Interferences, Fluorescent Signal of the Fluorogenic Dye, the RATIO at Time Zero of the Active Microbiological Fluorescent Signal of the Reacted Fluorogenic Dye to the Active Microbiological Fluorescent Signal of the Fluorogenic Dye and the RATIO at Time Zero of the Inactive Microbiological Fluorescent Signal of the Reacted Fluorescent Dye to the Inactive Microbiological Fluorescent Signal of the Fluorogenic Dye;
• (G) waiting for a time period, designated Time Future, and measuring the fluorescent signals in said Aliquot-Dye, said Optional Aliquot-Inhibitor-Dye, said Aliquot-Nutrient-Dye and said optional Aliquot-Blank at the wavelength of the Fluorogenic Dye and the Reacted Fluorogenic Dye at Time Future;
• (H) calculating the Useful RATIO at Time Future, wherein the Useful RATIO at Time Future is selected from the group consisting of RATIO at Time Future of the Total Microbiological, Optionally Accounting for Interactions with chemicals and Optionally Accounting for Background Interferences Fluorescent Signal of the Reacted Fluorogenic Dye to the Total Microbiological, Optionally Accounting for Interactions with chemicals and Optionally Accounting for Background Interferences, Fluorescent Signal of the Fluorogenic Dye, the RATIO at Time Future of the Active Microbiological Fluorescent Signal of the Reacted Fluorogenic Dye to the Active Microbiological Fluorescent Signal of the Fluorogenic Dye and the RATIO at Time Future of the Inactive Microbiological Fluorescent Signal of the Reacted Fluorescent Dye to the Inactive Microbiological Fluorescent Signal of the Fluorogenic Dye;
• (I) comparing the Useful RATIO at Time Future to the Useful RATIO at Time Zero;
• and
• (J) using the comparison of the Useful RATIO at Time Future to the Useful RATIO at Time Zero to monitor the extent of microbiological contamination in said opaque medium.